Structure-Function Studies of the Receptor Tyrosine Kinase MuSK

描述(申请人提供)：神经肌肉接头(NMJ)是一种高度专门化的突触，其形成依赖于穆斯克(肌肉特异性激酶)，穆斯克是一种受体酪氨酸激酶，由集聚蛋白激活，集聚蛋白是一种来自运动神经元的硫酸乙酰肝素蛋白多糖。麝香的激活导致肌肉蛋白重新分布到突触后部位，包括乙酰胆碱受体(AChR)、Rapsyn、ERBbs和麝香本身。此外，集聚蛋白诱导的麝香激活导致突触下核内突触特异基因的选择性转录上调，并诱导导致突触前分化的逆行信号。在成熟的NMJ中，运动神经元释放的乙酰胆碱激活AChRs并触发肌肉收缩。 麝香的胞外区和胞内区在导致NMJ形成的信号过程中都起着重要的作用。然而，这些信号转导过程背后的分子/结构机制尚不清楚。这个建议的目的是阐明控制麝香的结构机制：(I)集聚蛋白激活麝香；(Ii)麝香与AChR和Rapsyn共同聚集；以及(Iii)麝香重新招募非受体酪氨酸激酶Src和Abl。这项建议的具体目标是： 目的1.麝香胞外结构域的结构和功能特征 目的2.麝香与非受体酪氨酸激酶Src和Abl相互作用模式的分子表征 为了实现这些目标，我们将使用X射线结晶学来确定麝香胞外结构域和细胞质结构域的三维结构。然后，我们将通过在麝香缺失的肌管中表达特定的麝香突变体，并分析集聚蛋白诱导的麝香激活、AChR聚集以及Src和Ab1向麝香的招募，来表征从结构研究中确定的关键残基的功能作用。 相关性：NMJ的形成是一个基本的生物学过程，对生物体的发育至关重要。通过揭示这一重要突触形成的生化机制，我们将在分子水平上更好地理解这一过程中的缺陷是如何导致神经肌肉疾病的。

DESCRIPTION (provided by applicant): The neuromuscular junction (NMJ) is a highly specialized synapse whose formation is dependent on MuSK (muscle-specific kinase), a receptor tyrosine kinase that is activated by agrin, a heparan-sulfate proteoglycan derived from motor neurons. MuSK activation results in the redistribution of muscle proteins to the postsynaptic site, including the acetylcholine receptor (AChR), rapsyn, ErbBs and MuSK itself. In addition, agrin-induced MuSK activation leads to selective transcriptional upregulation of synapse-specific genes in subsynaptic nuclei, and to induction of a retrograde signal leading to presynaptic differentiation. In mature NMJs, acetylcholine released from motor neurons activates AChRs and triggers muscle contraction. Both the ectodomain and cytoplasmic domain of MuSK play essential roles in signaling processes leading to formation of the NMJ. However, the molecular/structural mechanisms underlying these signaling processes are not understood. The goals of this proposal are to elucidate the structural mechanisms governing: (i) MuSK activation by agrin; (ii) MuSK co-clustering with the AChR and rapsyn, and (iii) MuSK recruitment of the non-receptor tyrosine kinases Src and Abl. The specific aims of this proposal are: Aim 1. Structural and functional characterization of the MuSK ectodomain Aim 2. Molecular characterization of the modes of interaction between MuSK and the non-receptor tyrosine kinases Src and Abl To achieve these aims, we will employ x-ray crystallography to determine the three-dimensional structures of the MuSK ectodomain and cytoplasmic domain. We will then characterize the functional roles of key residues identified from the structural studies by expressing select MuSK mutants in MuSK-deficient myotubes and assaying for agrin-induced MuSK activation, AChR clustering, and recruitment of Src and Abl to MuSK. Relevance: Formation of the NMJ is a fundamental biological process which is critical for organismal development. By revealing the biochemical mechanisms underlying the formation of this important synapse, we will better understand, at a molecular level, how defects in this process give rise to neuromuscular disorders.